CD4 gene regulatory sequences specifically expressed in mature T cells

ABSTRACT

The present invention relates to compositions and methods for the expression of nucleic acids or polypeptides into mature T lymphocytes. The invention relates more specifically to methods of selective gene expression into mature T lymphocytes, based on CD4-derived regulatory sequences, such as CD4-derived enhancer sequences. The invention is particularly suited for regulating gene expression into mature T lymphocytes in vitro, ex vivo or in vivo, upon genetic modification of hematopoietic precursors and maturation thereof.

FIELD OF THE INVENTION

The present invention relates to the use of regulatory sequences whichare derived from the CD4 gene in vectors for expressing a heterologousgene or a transgene, with these sequences conferring on the vectors,when the latter are integrated into cells of the hematopoietic system,in particular stem cells, or, more generally, bone marrow cells, aspecific expression within mature T lymphocytes, excluding immature Tlymphocytes, in particular following repertoire selection.

BACKGROUND

During the maturation of T cells, expression of the CD4 and CD8receptors responds to a complex mechanism of regulation; differentialexpression of the CD4 and CD8 glycoproteins is coupled to the choice ofone of the pathways of differentiation either into helper T lymphocytesor into cytotoxic T lymphocytes; thus, thymocytes, that is thehematopoletic cells which are involved in a T differentiation pathway,first of all possess a CD4− CD8− phenotype (double negative or DNthymocytes); they then acquire joint expression of the CD4 molecule andthe CD8 molecule, thereby forming double positive (DP) CD4+ CD8+thymocytes and, finally, this population differentiates into singlepositive (SP) lymphocytes which express ether CD4, in the case of thehelper T lymphocytes, or CD8, in the case of the cytotoxic Tlymphocytes. Thymocytes which bind to class I histocompatibilitymolecules will become CD4+ CD8+ cytotoxic T lymphocytes while thosewhich bind to class II molecules will become CD4+ CD8− helper Tlymphocytes; after this intrathymic process of repertoire selection, thethyrnocytes leave the thymus and reach the peripheral system: i.e. bloodand lymphoid organs.

This differentiation process is summarized in Nicolic-Zugic, J, (1991)Immuncol. Today 12: 65-70.

Many groups are currently studying the regulation of the expression ofthe CD4 gene since elucidation of the mechanism of this expression couldcontribute towards understanding the manner in which T cell developmentis controlled.

Several groups are working on the regulatory sequences of the human ormurine CD4 gene. Those most recent studies which may be cited are thefollowing: Killeen et al. (EMBO Journal Vol. 12 No. 4 p. 1547, 1993)demonstrated that a transgene carrying human CD4 of a size ofapproximately 35 kb possessed all the requisite genomic sequences forcontrolling expression during development in transgenic mice. Twodistinct regulatory elements in this fragment were identified as beingcritical for expressing the transgene: the first is an enhancer sequencewhich is situated either 13 kb upstream of the cap site of the murineCD4 or 6 kb upstream of the human CD4. Blum M. D. et al. (J. Exp. Med.(1993) 177 No. 5: 1343-1358) identified and sequenced the human CD4enhancer while Sawada et al. (Mol. Cell. Biol; 11 55: 5506-5515, 1991)identified and sequenced the murine CD4 enhancer. These two groupsdemonstrated that expression of this enhancer is specific for the CD4gene in mature or immature T lymphocytes; P. Salmon et al. (Proc. Natl.Acad. Sci., USA 90: 7739-7743 (1993)) analyzed the structure and thesequence of the human CD4 promoter and compared it with that of themurine CD4 promoter. They identified a fragment of approximately 1100base pairs which exhibits the function of a specific CD4 promoter.Aligning this sequence with that of the murine CD4 promoter indicates avery similar structure, as FIG. 1 of this latter paper shows.

Differentiation of the T lymphocytes employs other regulatory elementsof the silencer type, whose presence leads to a decrease in, orcessation of, transcription of genes when they are in their vicinity. InCell 77: 911-929, (1994), Sawada et al. demonstrated the existence of asilencer in an initial intron of the CD4 gene, one of the functions ofwhich silencer is to extinguish transcription of the CD4 gene in matureCD8+ T lymphocytes.

Finally, the human CD4 gene was analyzed by Z. Hanna et al. (Mol. andCell. Biology (1994) p. 1084-1094) in a construct which comprises atleast 3 introns of a total length of 12 kb.

All the abovementioned recent studies indicate that expression of theCD4 cene is controlled in a similar manner in human and murine cells;nevertheless, the relationships between the different regulatoryelements and their functions have not been elucidated.

SUMMARY OF THE INVENTION

The present invention results from the discovery, which was made by theinventors, of a combination of regulatory sequences which exhibitproperties which are unexpected and entirely unforeseeable: thiscombination, which is composed of genetic elements which are derivedfrom sequences which are located 5′ of the CD4 gene, where appropriatecombined with the cDNA of the CD4 gene, is expressed in a restrictedmanner in mature T cells and NK cells in a transgenic mouse model; ithas not been possible to detect any expression in immature T cells,including double positive or double negative thymocytes. This group ofregulatory sequences is the first to have this specificity ofexpression, something which is especially important, in particular foruse in the context of gene therapy, for programs which would require agene to be expressed specifically in mature T lymphocytes.

There was nothing in the state of the art to suggest that it waspossible for such a combination to lead to this result.

The present invention results from the discovery that while thecombination of the CD4 enhancer and promoter promotes expression of areporter gene, in this case the cDNA of the human CD4 gene, in maturemouse thymocytes, this reporter gene is not expressed in immature CD4+CD8+ double positive thymocytes; this result is surprising in that itimplies the existence of an additional regulatory element which is asyet unknown and which would enable the CD4 gene to be expressed at theimmature stage; this element, which is thought to act in “cis”, isthought to be missing from the construct since no mouse regulatoryelement which would be able to act in trans is effective for bringingabout such expression in the immature cells.

A foreseeable result is that linking the silencer described by Sawada etal. 1994 (see above) to this combination forms a cassette for expressinga heterologous gene exclusively in CD4+ CD8− SP helper T cells.

The present invention relates to a system for expressing a protein or aheterologous gene, which system comprises a recombinant vector which canbe used for transducing cells of the hematopoietic line, in particularblood or marrow stein cells, in such a way that the cell which has beenthus transduced will only express the protein or the heterologous genewhich is carried by the vector in mature T cells after repertoireselection, to the exclusion of expression in immature T cells, inparticular CD4− CD8− and CD4+ CD8+ cells; said vector of the expressionsystem is provided with all the sequences which are required for itsexpression and it is characterized in that it contains at least oneenhancer of a CD4 gene which is derived from the same species or from adifferent species.

The vector of the expression system may also contain, combined with theenhancer, a promoter which consists of one of the following sequences:

the promoter of the human CD4 gene, as depicted in FIG. 6, or

the sequence contained between nucleotides −496 and +16 in the P. SALMON(1993, see above) numeration (FIG. 6), or

the sequence contained between nucleotides −165 and +16 (P. SALMON,1993, see above) (FIG. 6), or

any sequence which is derived from one of the preceding sequences bymeans of the addition, deletion or substitution of nucleotides withoutsubstantial modification of the expression of the heterologous proteinunder the control of this promoter.

A specific example of an efficient enhancer, within the context of thisinvention, is that of murine CD4, which enhancer consists of 339, or allor part of the 339 base pairs and is described in the abovementionedSawada et al. (1991), with it being possible for this enhancer to bepresent either in the form of a single sequence or in the form of asequence which is repeated from two to five times, and with the sequenceof this enhancer being the following (SEQ ID NO: 1:)

TGTTGGGGTT CAAATTTGAG CCCCAGCTGT TAGCCCTCTG CAAAGAAAAA AAAAAAAAAAAAAGAACAAA GGGCCTAGAT TTCCCTTCTG AGCCCCACCC TAAGATGAAG CCTCTTCTTTCAAGGGAGTG GGGTTGGGGT GGAGGCGGAT CCTGTCAGCT TTGCTCTCTC TGTGGCTGGCAGTTTCTCCA AAGGGTAACA GGTGTCAGCT GGCTGAGCCT AGGCTGAACC CTGAGACATGCATCCTCTGT CTTCTCATGG CTGGAGGCAG CCTTTGTAAG TCACAGAAAG TAGCTGAGGGGCTCTGGAAA AAAGACAGCC AGGGTGGAGG TAGATTGGT

The human CD4 enhancer described by M. D. Blum et al. (cited above)comes within the area of the invention, whether it is used alone or incombination with the CD4 promoter.

The term enhancer of a CD4 gene is understood as being not only thesequences described by Sawada et al. or by Blum et al. but also othersequences which are derived from other mammalian species and whichexhibit a sufficient degree of homology with the above sequences, withthese other sequences in particular including, for example, the specificCD4-1, CD4-2 and CD4-3 nuclear protein sequences described in Sawada etal. (1991).

The implication of this specific property—i.e. non-expression inimmature T cells and expression in mature T cells—reveals the advantagesof incorporating the construct of the invention into medicaments whichcan be used in gene therapy for all the indications where transgeneexpression in T cells could be a means of treating or preventing certainpathologies or dysfunctions, in particular viral infections,particularly infections due to HIV, pathologies in the expression ofcertain genes in the T lymphocytes, for example deficiency in adenosinedeaminase or, more generally, primary or secondary immune deficiencies,autoimmune pathologies or grafts. However, the therapist comes upagainst the following difficulty: if mature T lymphocytes are taken asthe targets for gene therapy, he is obliged to transfer genes into thesemature T lymphocytes ex vivo and these latter lymphocytes have to bepreviously cultured in order to effect this transfer using theappropriate vectors; after reinjection, the half-life of the Tlymphocytes is relatively short; by contrast, if the strategy selectedis to target the hematopoietic stem cells which give rise, afterdifferentiation, to the T lymphocytes, two different problems are thenmet with: the first is that of an expression of the gene of interest inthe stem cells which is not always desirable, and the second is that ofthe possible disruption of repertoire selection in the thymus which isinduced by expression of the heterologous gene in immature cells.

The construct of the invention enables this double drawback to beovercome by means of transferring, to the hematopoietic stem cells, aheterologous gene which will only be expressed after the repertoireselection has taken place.

As has been mentioned above, the transfer of genes into hematopoieticcells has been envisaged for expressing a protein of interest of theadenosine deaminase type in mature T lymphocytes for which the currenttreatment is transduction of cultured T lymphocytes, which have beenharvested by means of leucopheresis, with a recombinant retroviralvector (for a summary of these experiments, see Anderson W. F. HumanGene Therapy, Science (1992), 256: 803-814).

Serious viral infections (AIDS) or the prevention of immune stimulationof the autoimmune disease or GVHD (graft-versus-host disease) typerepresent another type of application of the transfer of genes intohematopoietic cells. The approach consists in booby-trapping Tlymphocytes by incorporating into them a transgene which consists of asuicide gene, for example HSV1 thymidine kinase (HSV1-TK) or afunctional equivalent of this gene whose expression product is able tometabolize, in situ, a pharmaceutically inactive substance into a toxicderivative for specifically inducing the destruction of these cells,with an example of such a derivative in the case of HSV1-TK beingganciclovir.

Obtaining expression vectors which possess the specific expressionqualities of the vectors of the invention makes it possible to overcomethe abovementioned drawbacks of the transfers into stem cells or intomature cells.

The vectors which can be used for transferring genes into hematopoieticstem cells (HSCs), and which are able to incorporate the regulatoryelements of the invention which enable the foreign gene to bespecifically expressed exclusively in mature cells, may be selected fromany of the vectors which are suitable for transfecting these HSCs. Thesevectors are essentially viral vectors of the adenovirus oradenoassociated virus (AAV) type or retroviral vectors, or elsenon-biological systems for delivering nucleic acid in vivo. A summary ofthe essential characteristics of these different types of vector will befound in “Thérapie génique (Gene therapy), Edition John Libbey, (1993)pages 3 to 33”.

Specific retroviral vectors have been described for booby-trapping Tcells either with regard to infection with retroviruses or in order toinduce GVHD tolerance and are described in U.S. Pat. Nos. 6,048,525 ,5,843,432 and U.S. Pat. No. 5,948,675, and patent application No.WO93/08844 the contents of which patent applications are incorporatedinto the present application by reference.

In the expression system of the invention, whose expression vectorcontains a cassette which consists of the above-described promoter orenhancer/promoter combination, the heterologous protein which is capableof being expressed in mature T lymphocytes can be selected as a proteinwhich exhibits a toxicity which is dependent on the presence of asubstance: thus the heterologous protein can advantageously be HSV1-TKthymidine kinase or a protein which is derived from this thymidinekinase as long as it retains the functional characteristics of a kinase;while the substance can be a nucleoside analog such as acyclovir organciclovir, which is capable, after having been phosphorylated by thethymidine kinase, of being incorporated into dividing DNA and ofbringing about the death of the mature T lymphocyte.

Examples of constructs which enable this “suicide gene” approach to beimplemented in CD4+ T lymphocytes by means of transfecting an HSV1-TKgene and treating with ganciclovir have been described in Caruso M. andKlatzmann D. (Proc. Natl. Acad. Sciences (1992) 89: 182-186).

The present invention also relates to cells of the hematopoietic cellline, in particular HSCs which are transfected with an expression vectorwhich consists at least of a sequence encoding a heterologous protein, apromoter and a polyadenylation sequence, which cells are characterizedin that the said vector contains at least one enhancer sequence from amammalian CD4 gene of the same species or of a different species, whereappropriate in combination with a CD4 gene promoter, with theheterologous protein being principally expressed in mature T lymphocyteswhich are derived from repertoire selection.

Preferably, the promoter consists of one of the above-describedsequences, and consists, or is derived from, the human CD4 gene promoteras depicted in FIG. 6.

The promoter can also be a cytokine promoter or the promoter of acytokine receptor such as that of interleukin 2.

In the expression systems of the invention, the expression vectorcontains, upstream or downstream of the sequence to be expressed, amurine CD4 enhancer which consists of the 339 base pair sequencedescribed in Sawada et al. (1991). One embodiment is to include between1 and 5 copies of this enhancer or of a derived enhancer as definedabove, preferably 3 copies.

The cells which are transfected with a recombinant vector expressing aprotein which exhibits conditional toxicity for mature T cells also formpart of the invention; in particular, cells which are transfected with avector carrying HSV1-TK thymidine kinase or a protein which is derivedfrom this kinase forms part of the invention. The thymidine kinase genecan then be dependent on a promoter such as described above or on aspecific promoter, in particular the interleukin 2 promoter, which iscombined in any manner with an enhancer as described above. It should berecalled at this point that activation in the case of T lymphocytes isassociated with cell division; since the ganciclovir is only toxic forcells which are dividing and expressing HSV1-TK, it is not, therefore,necessary to be able to control expression of the HSV1-TK gene extremelystrictly during cell activation. An expression which is restricted tomature T lymphocytes is adequate; thus, even if a quiescent mature Tlymphocyte expresses the HSV1-TK protein constitutively, this lymphocytewill not be destroyed, even in the presence of ganciclovir. By contrast,as soon as the cell has been activated and is dividing, the ganciclovirtriphosphate will already be present in the cell and will make itpossible to eliminate the cell immediately.

The therapeutic strategy which consists in carrying out a bone marrowgraft using cells which harbor an HSV1-TK transgene under the control ofthe regulatory sequences of the invention will then afford the requisitesecurity of the gene not being expressed during the whole of the Tlymphocyte differentiation process and therefore of not disruptingrepertoire selection and of not risking the possibility of killing cellswhich are in permanent division, as the immature thymocytes are.Finally, it is a sought-after aim that the TK gene should be expressedpermanently by all the T lymphocytes. If a graft-versus-host reactionappears when bone marrow stem cells which are transduced with TK arereinjected into a patient, with the graft-versus-host reaction involvingactivation of certain T lymphocytes of the graft which recognizehistocompatibility antigens of the recipient, the TK system should makeit possible to eliminate these lymphocytes by treating the patient withganciclovir or acyclovir.

The present invention also relates to the use of specific regulatorysequences of the CD4 gene, in particular the enhancer, or thecombination of the enhancer and a promoter, as described above, in theproduction of a medicament which can be used in gene therapy forspecifically destroying activated T lymphocytes, with these regulatoryelements being integrated into an expression system which is capable oftransforming the cells of the hermatopoietic system; such a use makes itpossible to envisage specifically expressing the transgene, which hasbeen incorporated into the vector, within mature CD4+ CD8− and CD4− CD8+T cells, with this expression being totally repressed during the entiredifferentiation process up to and including repertoire selection in thethymus.

We recalled above that Sawada et al. demonstrated, in Cell (1991), theexistence of a transcriptional silencer which is specific for the CD4gene and which is located in the first intron of the gene. Thecombination, which has thus been characterized, of the silencer and theregulatory sequences of the invention, whether they be the enhanceralone or the enhancer combined with a promoter, with enhancer andpromoter retaining the above definitions, form part of the invention asdoes their use in the production of a medicament which can be used ingene therapy for transforming the stem cells of the hematopoieticsystem. Such a combination of regulatory elements in a vector forexpressing a heterologous protein makes it possible to envisageexpressing this heterologous protein exclusively in mature CD4+ CD8− Tlymphocytes, excluding immature T lymphocytes and CD8+ cytotoxic Tlymphocytes.

The use of an expression system which comprises vectors which areprovided with the regulatory sequences of the invention is particularlyrecommended in the production of a gene therapy medicament forpreventing or treating graft rejections or graft-versus-host reactionsor for treating autoimmune diseases; it can also be taken intoconsideration for preparing a medicament which can be used forpreventing or treating infections due to viruses, in particular due toHIV. The use of the construct which additionally comprises a silencercan make it possible to prepare medicaments for gene therapy.

Thus, the findings which are currently available suggest that thecombination enhancer/promoter/silencer of the CD4 gene should besufficient to obtain expression in all CD4+ cells (including DP cells).However, as reported in this invention, it appears that theenhancer/promoter combination clearly has an innovative character sinceit brings about expression which is restricted to mature T cells; whilethe absence of the silencer only has an effect on expression in CD8+cells, it is not linked to the stage of development.

The examples below illustrate the unexpected characteristics of thecombination of regulatory elements of the invention and the propertieswhich it confers on the cells into which the elements have beenintegrated.

On the basis of the findings which are described in this document, theskilled person will very readily know how then to adapt the constructwhich should be employed for a specific prophylaxis or therapy which hemight wish to implement and which requires specific expression in matureCD4+ CD8− and CD4− CD8+ T cells or exclusively in mature CD4+ CD8− Tcells.

BRIEF DESCRIPTION OF THE DRAWINGS

In the examples which follow, the figures have the following legends:

FIG. 1A depicts the pCD4 cassette, which consists of the 1100 base pairhCD4 promoter, the human CD4 cDNA, which is used here as a reportergene, and the SV40 virus polyadenylation sequence.

FIG. 1B depicts the EpCD4 cassette, which consists of the same elementsas in the case of pCD4, which elements are provided upstream with 3copies of the 339 base pair sequence of the mouse CD4 enhancer.

In FIGS. 1A and 1B, the letters indicate restriction sites and have thefollowing meanings: H=Hind3, S=Sph1, P=Pst1, X=Xba1, B=BamH1, E=EcoR1.

FIG. 2 depicts the expression of the hCD4 reporter gene at the surfaceof mononuclear cells from transgenic mouse spleen, i.e. SP CD4+, SPCD8+, γδ lymphocytes, B lymphocytes, macrophages and NK cells,respectively.

FIG. 3 depicts an analysis of the expression of the hCD4 reporter genein transgenic mouse thymocytes by means of triple immunolabeling andflow cytometry. (A) shows the distribution of the thymocytesubpopulations which are defined by the expression of mCD4 and mCD8. Thenumbers in each field represent the percentage of each subpopulation inthe thymus. (B) shows hCD4 expression in DN (CD4− CD8−); DP (CD4− CD8+);SP (CD4+ CD8−) and SP (CD4+ CD8+) cells, respectively.

The numbers represent the percentage of hCD4+ thymocytes in eachsubpopulation for the two experiments which are depicted in this figure.Each result was reproduced seven (line 7) and thirteen (line 10) timesin independent experiments.

FIG. 4 depicts an analysis of the expression of CD3, TCR αβ and TCR γδin DN thymocytes which are expressing hCD4. The transgenic mousethymocytes were labeled with a QR-coupled anti-hCD4 monoclonal antibody,a mixture of PE-coupled anti-mCD4 and anti-mCD8 antibodies and either(A) an FITC-coupled anti-TCR αβ antibody or an FITC-coupled anti-TCR γδantibody. The phenotypes of the DN thymocytes (A) and the DN thymocytesexpressing hCD4 (B) were studied by making windows on the PE− cells andQR+PE− cells, respectively. The results depicted in this figure arerepresentative of five (line 10) and one (line 7) reproducibleexperiments.

FIG. 5 depicts an analysis of the expression of hCD4 and HSA on SP CD4thymocytes. The SP CD4 thymocytes were isolated by cell-sorting andwashed; they were then subjected to a second immunolabeling withPE-coupled anti-HSA and QR-coupled anti-hCD4 monoclonal antibodies. Theexperiment which is depicted was reproduced three (line 10) and one(line 7) times in independent experiments.

FIG. 6 (SEQ ID NO:2) depicts the sequence of the promoter of the geneencoding human CD4 and is taken from P. Salmon et al. (Proc. Natl. Acad.Sci., USA 90: 7739-7743 (1993)).

FIG. 7 depicts the characterization of the human CD4 gene silencer (Sil)which makes it possible to express a transgene differentially in matureCD4+ T lymphocytes and not in mature CD8+ lymphocytes.

FIG. 8 depicts the specific depletion, by ganciclovir, of CD4+ and CD8+T lymphocytes (FIG. 8a) in response to a mitogen (ConA) in vitro and(FIG. 8b) the in-vivo depletion of T lymphocytes expressing TCR Vβ7 inresponse to stimulation with the superantigen SEB.

FIG. 9 shows the results of an analysis of irradiated mice which aregiven a bone marrow graft which is or is not accompanied by splenocyteswhich are derived either from transgenic mice or from normal mice. Thecontrol mice, which are not grafted (BMG−) die as a consequence of theirradiation. The animals which are grafted with bone marrow alone,without splenocytes (BMG+), survive due to the bone marrow graft andthere is no GVH as is the usual case in mice. The animals which aregiven a bone marrow graft and allogenic splenocytes which are derivedeither from transgenic mice which are treated with PBS (GVH/PBS) or fromnon-transgenic mice which are treated with ganciclovir (GVH/GCV NTG) dieequally rapidly and exhibit all the clinical and histological signs of aGVH. Only the mice which have been given the bone marrow and splenocytesfrom transgenic mice which have been treated with ganciclovir (GVH/GCV)survive in the same way as the mice given the marrow graft, without anyhistological or clinical signs of GVH. The BMG− group is represented bycurve A. The BMG+ group is represented by curve B. The GVH/GCVNTG groupis represented by curve C. The GVH/PBS group is represented by curve Dand the GVH/GCV group is represented by curve E.

FIG. 10 depicts the characterization of the minimum sequences, derivedfrom the regulatory sequences of the human CD4 gene, which are requiredfor expressing a transgene. The transcription initiation site of thehuman CD4 gene is represented by a black arrow. The (reporter) gene forsecreted human alkaline phosphatase is embodied by the acronym pSEAP.The black and gray-tinted rectangles represent the conserved 2 “box” and3 “box” mouse-homologous sequences, respectively. Finally, “MEH3X”corresponds to the murine CD4 gene enhancer which, in the present test,is repeated three times.

FIG. 11 depicts expression of the reporter gene following transfectionof the different gene constructs of line 4 into Jurkat cells.

DETAILED DESCRIPTION EXAMPLE 1

Expression of a Human CD4 Minigene in the Mature T Cells of TransgenicMice:

Two plasmids were constructed:

the plasmid pCD4, which carries a 1100 base pair sequence correspondingto the human CD4 promoter and, downstream of that, the human CD4 cDNAand an SV40 polyadenylation sequence (FIG. 1A),

the plasmid EpCD4, which is the same construct but which additionallycomprises three repeats of the 339 base pair murine CD4 enhancerdescribed in Sawada (1991) (FIG. 1B).

The CD4 cDNA is included in these constructs as a reporter gene, that iswhose expression or non-expression in the transgenic mouse system isobserved, with the expression being easy to detect by means of the flowcytometry techniques employed.

1—Materials and Methods

1a—Construction of the Transgenes

In order to construct pCD4, depicted in FIG. 1A, the Pst1 fragment ofthe human CD4 (hCD4) promoter described by P. Salmon et al. (1993) andcomprising nucleotides −1076 to +20, was ligated to the 1.8 kbEcoR1-BamH1 cDNA fragment encoding the human CD4 protein; this proteinhas been characterized by Maddon P. J. et al. in Cell 42: 93:104 (1985).The polyadenylation sequence is derived from an 0.8 kb BamH1-Bgl2fragment from the SV40 virus.

In constructing plasmid EpCD4 (FIG. 1B), a PCR-amplified fragment of themurine CD4 enhancer was added to the pCD4 construct directly upstream ofthe hCD4 promoter. Briefly, murine genomic DNA was subjected to PCRamplification using primers which were complementary to the flankingsequences of the 339 base pair minimum sequence of the above-describedenhancer, which sequence contains additional Sph1 restriction sites. ThePCR products were cloned into plasmid pCD4 and the sequences of theinserts were verified. A clone containing 3 copies of the murineenhancer was retained for constructing transgenic mice.

1b—Generation and Screening of the Transgenic Mice

Hind3-EcoR1 DNA fragments were generated from plasmids pCD4 and EpCD4and separated by means of agarose gel electrophoresis. The transgeneswere then eluted using the BIOTRAP™ elution system (Schleicher andSchuell, Dassel, Germany) and adjusted to a concentration of 5 ng/μl in10 mM Tris-HCl, pH 7.4, 0.1 mM EDTA.

Transgenic mice harboring plasmids pCD4 and EpCD4 were obtained andcharacterized as described by Hogan B. et al. (1986) in Manipulating theMouse Embryo, a Laboratory Manual; Cold Spring Harbour Laboratory Press,Cold Spring Harbour, N.Y. The transgenes were microinjected into thepronuclei of F2 zygotes which were obtained by crossing two: [C57 B1/6DBA/2] F1s. The transgenic animals were detected by hybridizing DNAprepared from tail biopsies with a radiolabeled probe which is specificfor human CD4 and which is the 1.8 kb EcoR1-BamH1 fragment of human CD4and is described in the article by Maddon P. et al. (1985).

1c—Antibodies Employed

The following directly labeled monoclonal antibodies were employed, withthe initials having the following meanings: FITC=fluoresceinisothiocyanate, PE: phycoerythrin; QR: quantum red; SA: streptavidin;APC: allophycocyanin; PerCP: peridinine chlorophyll protein; m: murine;h: human.

List of the antibodies:

anti-mCD4 (rat IgG2b labeled with PFE or APC, clone YTS 191.1),

anti-mCD8 (rat IgG2b labeled with FITC or PE, clone YTS 169.4),

anti-mCD3 (hamster IgG labeled with FITC, clone 500-A2),

anti-mB220 (rat IgG2a labeled with FITC, clone RA3-6B2),

anti-mTCRαβ (hamster IgG labeled with FITC, clone H57-597) and

anti-mMAC1 (rat IgG2b labeled with FITC, clone M1-70.15),

obtained from Caltag Laboratories (San Francisco, Calif.);

anti-mTCRγδ (hamster IgG labeled with PE, clone GL3),

anti-mNK (mouse IgG2a labeled with PE, clones PK 136 and 5E6),

anti-mHSA (rat IgG2b labeled with PE, clone M1/69) obtained fromPharMingen (San Diego, Calif.);

anti-hCD4 (mouse IgG1 labeled with QR, clone Q4120) and a negativecontrol (mouse IgG1 labeled with QR, MPOC-21)

obtained from Sigma Immunochemicals (St. Louis, Mo.)

1d—Preparation of the Cells and Analysis by Flow Cytometry

All the analyses were carried out on mice of from 8 to 20 weeks of age.The thymus, the spleen and the lymphatic ganglia are disrupted in DMEM(Dulbecco's modified Eagle medium) obtained from Life Technologies Inc.(Gaithersburg, Md.), resuspended with a pipette, centrifuged at 1100 rpmfor 5 minutes and then resuspended in a labeling buffer consisting ofPBS containing 2% fetal calf serum and 0.02% sodium azide. Thesuspension of splenic cells was mixed with two volumes of 0.8% ammoniumchloride in order to lyse the erythrocytes and was then immediatelycentrifuged and resuspended in the labeling buffer. After counting, allthe cells in suspension are adjusted to a final concentration of from 10to 20×10⁶ cells/ml. For analyzing the B cells and the macrophages, afinal concentration of 2% autologous serum was added to the colorationbuffer before adding the monoclonal antibodies. The labelings werecarried out by incubating from 1 to 2×10⁶ cells with the monoclonalantibodies at 4° C. for 30 minutes; after a final wash, the cells werefixed in from 0.5 to 1 ml of 1% paraformaldehyde in PBS. From 10 to40,000 cells were analyzed by flow cytometry (FACSCAN™, BectonDickinson, San Jose, Calif.) in this way.

Purified SP CD4+ CD8− thymocytes were obtained after subjecting a totalthymus cell suspension to double color-labeling with APC-labeledanti-mCD4 antibodies and PE-labeled anti-mCD8 antibodies for 1 hour inice and were then washed and filtered through a FALCON 2350™ (BectonDickinson, Franklin Lakes, N.J.) nylon cell filter. The APC+ and PE−cells are sorted, in PBS buffer containing 0.02% sodium azide, using aFACSTARPLUS™ (Becton Dickinson).

When 10⁶ SP CD4+ CD8− cells have been obtained by cell-sorting, they arewashed and resuspended in 250 μl of labeling buffer; they are thenseparated into two tubes and subjected to labeling with PE-labeledanti-HSA antibodies and either QR-labeled anti-hCD4 antibodies or withisotype control antibodies. The cells were analyzed on a FACSCAN™ asdescribed above.

2—Results

2-1 Detectable Expression of the CD4 cDNA Reporter Gene in Vivo Requiresthe Presence of the Enhancer in Addition to the CD4 Promoter.

Table 1 below summarizes the percentage of cells expressing the hCD4reporter gene in the thymus, the spleen, the lymphatic ganglia and thePBMNCs (peripheral blood mononuclear cells); the presence of the CD4 wasanalyzed by means of flow cytometry as described above. The figures inbrackets indicate the number of animals tested, while the results arethe means obtained for the different animals tested plus or minus thestandard deviation.

TABLE 1 Lymphatic Construct Thymus Spleen ganglia PBMNCs pCD4 (7 cell <1<1 N.D. <1 lines) EpCD4 8 ± 3.0 (10) 30 ± 6.1 (10) 75 ± 13.4 (6) 58 ±14.0 (6)

Seven transgenic lines were obtained which harbored from 1 to 40 copiesof the pCD4 plasmid, which latter did not, therefore, include theenhancer; it was not possible to detect any hCD4-positive cell underthese conditions.

In the case of the EpCD4 construct, four lines were obtained harboringfrom 5 to 20 copies of the construct, all of which lines express thehCD4 transgene; two of the lines were analyzed in detail (line 7 andline 10). The results for EpCD4 which are hown in the table wereobtained with line 10, while similar results were obtained with line 7.

The cells of the spleen, the lymphatic ganglia and the PBMNCs are maturecells obtained after repertoire selection; this explains the markedlygreater percentage of cells expressing CD4 in these categories ascompared with the percentage obtained for the thymus.

In conclusion, it appears that the presence of the CD4 gene enhancer isnecessary and sufficient for obtaining expression of the reporter gene.

2.2—Expression of the EpCD4 Transgene in Peripheral Lymphoid Organs.

The transgenic EpCD4 Lines 10 and 7 were analyzed in order to determinethe cell types expressing the transgene. Thus, we carried out flowcytometry analyses after doubly or triply labeling splenocytes usingmonoclonal antibodies against human CD4 in combination with monoclonalantibodies which recognized different hematopoietic cell types.

As FIG. 2 shows, the expression of hCD4 is restricted to T lymphocytesand to NK cells. Expression of hCD4 was detected in all the T cells atthe same level as the cells express CD4 or CD8. Comparable expression isobtained in lymphocyte cells expressing a γδ receptor. It was notpossible to detect any expression in either the monocytes or the Blymphocytes of the mice which were analyzed (FIGS. 2D-E).

The majority of the NK cells, defined as being recognized by both thePK136 and 5E6 monoclonal antibodies, were also found to express hCD4 tovariable extents (FIG. 2F). Taken as a whole, these results show thatexpression of the EpCD4 transgene in peripheral cells is restricted to Tcells and to NK cells.

The results shown in this figure were reproduced from three to twelvetimes in independent experiments. Two different monoclonal antibodies(PK136 and 5E6) were used in the analysis of the NK cells, with theseantibodies revealing a proportion of hCD4+ cells of from 74 to 100%.These frequencies are always slightly higher in the 5E6+ cells than inthe PK136+ cells.

The results which were obtained for the SP CD4+ and SP CD8+ lymphocyteswere duplicated in the case of the lymphatic ganglia and the peripheralblood mononuclear cells.

While these results were obtained with line 10, comparable results wereobtained with line 7.

3—Expression of the EpCD4 Transgene in Thymocytes.

In FIG. 3, which depicts an analysis of the expression of the hCD4reporter gene, the mean frequencies of hCD4+ thymocytes found in DN, DP,SP CD4 and SP CD8 cells are (mean plus or minus standard deviation): 21%plus or minus 6.7%, less than 1%, 62% plus or minus 8.4% and 67%. plusor minus 18.1% for line 10 (n=13), and 13% plus or minus 6.0%, less than1%, 41% plus or minus 12.9% and 68% plus or minus 13.0% for line 7(n=7). While hCD4 cannot be detected in the DP thymocytes, a verysignificant fraction of the SP thymocytes expresses the transgene.

Expression of hCD4 was detected in a minor fraction of the DN thymocytes(from 10 to 25% on average). Although these DN and hCD4+ cells representless than 1% of the total thymocytes, it was important to verify theirphenotype in order to determine whether they were precocious DN thymicprecursors or mature DN T cells (in that case expressing a TCR/CD3). Forthis, a triple labeling was carried out using:

a mixture of PE-labeled anti-CD4 and anti-CD8 antibodies,

a QR-labeled anti-hCD4 antibody,

either an FITC-labeled anti-CD3 antibody, or an FITC-labeled anti-TCR αβantibody or an FITC-labeled anti-TCR γδ antibody.

As FIG. 4 shows, the double negative hCD4+ thymocytes all consist ofcells which are expressing a CD3/TCR receptor; of these cells, ¾ areexpressing TCR αβ while ¼ are expressing TCR γδ. This therefore meansthat the EpCD4 transgene is not expressed in immature DN thymocytes butin mature DN thymocytes which are expressing an antigen receptor.

By contrast, the EpCD4 transgene was found to be expressed in a verysubstantial fraction of the SP thymocytes, whether their phenotype wasCD4+ or CD8+ (FIG. 3).

These results are very surprising and it was not possible to predictthem on the basis of currently available information.

From the above it follows that the CD4 minigene is expressed in allperipheral T cells and only on a fraction of the SP thymocytes; thequestion therefore presents itself of knowing whether this expression inthe SP thymocytes correlates with the stages of SP thymocytedifferentiation.

In order to answer this question, SP CD4+ CD8− thymocytes were analyzedfor the expression of HSA and hCD4 at their surface. This experiment isdepicted in FIG. 5 and shows clearly that only the most mature (HSA−)thymocytes express hCD4, while their HSA+ precursors do not express itand there therefore exists a line of descent from hCD4− HSA+ to hCD4+HSA− in the SP CD4+ thymocytes.

These results demonstrate that expression of the EpCD4 transgene isdirectly linked to the stage of maturation of the T cells, whether thecell line is CD4+ or CD8+: the expression appears on the most mature SPthymocytes and then persists peripherally.

All in all, this combination of regulatory sequences (promoter+enhancer)controls expression of a reporter gene in only mature T cells and avariable proportion of NK cells. This is an observation which was notforeseeable since previously available information suggested that thecombination of these elements would control the expression of a reportergene from the DP stage onwards. They imply the existence of anadditional, as yet unidentified, regulatory element which makes itpossible to obtain expression in DP thymocytes and which could bepresent in the first intron of the CD4 gene.

EXAMPLE 2

Expression of an HSV1-TK Minigene in Mature T Cells from TransgenicMice:

An EpTK plasmid was constructed; it consists of the same regulatoryelements as the previously described EpCD4, that is 3 copies of themurine enhancer, the proT4 human CD4 promoter and the SV40 PolyA signal.It differs from EpCD4 in that the hCD4 cDNA has been replaced with a 1.3kb fragment containing the DNA encoding HSV1-TK. Transgenic mice wereprepared for the purpose of specifically destroying dividing (activated)mature CD4+ and CD8+ T lymphocytes by means of treating withganciclovir. This destruction also affects DN CD3+ cells, γδ lymphocytesand a proportion of NK cells. On the other hand, this destruction doesnot affect immature DP thymocyte precursors, which represent theprincipal population of the thymus. The T lymphocytes which come fromthe lymphatic ganglia of the transgenic mice are destroyed in vitro byganciclovir when they are cultured in the presence of a mitogen such asconcanavalin A.

A progressive disappearance of a proportion of mature T lymphocytes overtime is observed in these transgenic mice which are being used forstudying homeostasis and the rate of T lymphocyte renewal when the miceare treated with ganciclovir.

When these transgenic mice suffer an induced deletion of the lymphocyteswhich respond to a given antigen, a specific antigen tolerance isobtained when ganciclovir is administered in the period surroundingimmunization with the antigen.

In these transgenic mice, a graft-versus-host reaction is seen to becontrolled during treatment with ganciclovir when this reaction has beeninduced by a bone marrow graft which is mixed with mature T lymphocytesfrom EpTK mice. Another result of this is treatment or prevention of thegraft-versus-host reaction while preserving the graft-versus-leukemia(GVL) reaction.

In transgenic mice which have been prepared using EpTK-type constructswhich have been modified by adding various previously describedregulatory elements derived from the human CD4 gene or from CD4 genes ofother species, in particular the silencer of the CD4 gene in CD8lymphocytes and various sequences derived from the first intron of theCD4 gene, T cell populations in which HSV1-TK is expressed when thesecell populations are dividing are destroyed by ganciclovir.

Different constructs which contain HSV1-TK and which are placed underthe control of the previously described regulatory sequences derivedfrom the CD4 gene of various animal species can be prepared with the aimof producing expression vectors. These vectors are, on the one hand,non-viral vectors and, on the other hand, viral vectors, in particularretroviral vectors or vectors which are derived from AAV or adenovirus.Any of these different vectors can be used for transducing the HSV1-TKgene under control of the various previously described regulatorysequences either into peripheral T lymphocytes which are cultured exvivo or into bone marrow cells, in particular hematopoietic stem cells.An expected result is the duplication of the specificity of expressionwhich was obtained with the transgenic mice expressing HSV1-TK under thecontrol of the same regulatory sequences as those of the vector, eitherafter its transduction into the peripheral T lymphocytes ex vivo orafter its transduction into the hematopoietic stem cells.

EXAMPLE 3

Gene Construct Which Enables Specific Expression to be Obtained inMature CD4+ T Lymphocytes:

The addition of an, already identified, so-called “CD4 silencer”sequence to the construct containing the CD4 promoter and enhancer makesit possible to prepare a gene construct whose expression is nowrestricted exclusively to mature CD4+ T lymphocytes in the transgenicmice (FIG. 7). Double negative thymocytes, double positive thymocytesand mature CD8+ T lymphocytes do not express the transgene. As aconsequence, this type of construct can be used to transport a transgeneexclusively into mature CD4+ T lymphocytes, for example with a view tocarrying out a gene therapy, when these regulatory sequences areincluded in a retroviral vector.

Deletion of Specific T Cell Clones:

The CD4 promoter and CD4 enhancer regulatory sequences which werepreviously used in the transgenic mice for achieving exclusiveexpression in mature CD4+ or CD8+ T lymphocytes were used to express anHSV1 TK suicide gene. Transgenic mice were prepared using this geneconstruct.

The functionality of the gene construct is demonstrated by culturingmature lymphocytes from the ganglia of these transgenic mice in thepresence of ganciclovir after having activated the lymphocytes in vitrowith a mitogen. The concentrations of ganciclovir which are toxic forthe transgenic mouse cells are 100 times lower than those which arerequired to kill T lymphocytes from non-transgenic mice (FIG. 8a).

With this functionality having been demonstrated, the use fortherapeutic applications of expressing an HSV1-TK gene under the controlof these regulatory sequences can then be analyzed. The general idea isto destroy the cells when they are activated by the antigen insituations where T lymphocytes are responsible for pathologies. Thefeasibility of this procedure can be demonstrated by injecting asuper-antigen which is known to specifically activate lymphocytescarrying a Vβ7 into transgenic mice and control mice. In either the CD4+or the CD8+ populations of the control mice, activation with thesuperantigen SEB results in a doubling of the percentage of the Vβ7cells. By contrast, when the mice are treated with ganciclovir, there isno change in the number of these cells (FIG. 8b). These resultsdemonstrate that it is possible specifically to destroy cells which areactivated by an antigen when the T lymphocytes express the HSV1-TK geneunder the control of these regulatory sequences.

Treatment of the Graft-versus-host Reaction:

The functionality of this system for controlling a graft-versus-hostreaction was tested. Irradiated mice are reconstituted with bone marrowcells and splenocytes derived from transgenic mice which are expressingthe HSV1-TK gene under the control of the CD4 promoter and the CD4enhancers. This marrow graft is carried out in an allogenic context andunder this situation, the splenocytes which are reinjected at the sametime as the bone marrow are responsible for a fatal graft-versus-hostreaction.

In these experiments, 100% mortality is observed in animals which havereceived such a marrow graft under allogenic conditions. By contrast,treatment with ganciclovir for 7 days following the marrow graft issufficient to prevent the development of a graft-versus-host reactioncompletely. Under these conditions, most of the animals survive themarrow graft without any sign of GVH (FIG. 9).

The table below shows percentage survival in transgenic TK mice based onthe results of 3 experiments which lasted from 120 d to 41 d and whichwere carried out on irradiated mice:

TABLE 2 CONTROL TREATED GROUPS BMG+ ANIMALS ANIMALS 84% 0% 93% (n = 13)(n = 23) (n = 14)

The above table shows that while the irradiated mice which are given abone marrow graft (BMG+) survive, the same mice which are given bonemarrow and splenocytes die of GVH (control) unless the splenocytes arederived from transgenic mice and the mice are treated with ganciclovir(treated animals).

The demonstration that this phenomenon is indeed due to destruction ofthe T lymphocyte clones which are involved in the GVH can be provided bystudying the functionality of the lymphocytes in these mice. Thus, whenthese lymphocytes are withdrawn and activated in mixed lymphocytereactions either using lymphocytes of the same origin as the recipientor lymphocytes of a third-party origin as the stimulatory cells, it isobserved that while there is normal activation against the third-partycells, there is no activation against the recipient cells. These resultsdemonstrate that clones which are specifically capable of recognizingthe alloantigens of the recipient, and which are involved in the GVH,have been successfully deleted from the mice which survived the marrowgraft.

EXAMPLE 4

Constructs which Contain Different Sequences of the Human CD4 Promoter:

Different gene constructs were prepared in order to ascertain theminimum regulatory sequences, derived from the CD4 gene sequences, whichare required for expressing a transgene specifically in T lymphocytes.All these constructs are used for controlling the expression of areporter gene P. The basic construct contains the 1100 base pairs of theCD4 gene promoter and the CD4 enhancer, which is present in 3 copies.Starting with this construct, other different gene constructs wereprepared in accordance with the scheme shown in FIG. 10.

The results of analyzing the expression of the reporter gene aftertransfecting these different gene constructs of line 4 into Jurkat cellsdemonstrate (FIG. 11) that:

1) in a general manner, expression is 10 times greater in the presenceof the CD4 enhancer than in its absence;

2) the −169+16 fragment of the Pβ1-2C construct is as efficacious as thewhole of the 1100 base pair fragment in directing expression of thereporter gene.

In conclusion, these results demonstrate that the short regulatorysequences which are derived from the CD4 gene promoter and enhancer, andwhich are of a size which is compatible with their use within viralvectors such as retroviral vectors, can be used to obtain specificexpression in T lymphocytes.

The same constructs which are derived from CD4 regulatory sequences arenot expressed in non-lymphoid CD4+ cells such as, for example, HELAcells.

These experiments also demonstrate that the presence of a single copy ofthe CD4 enhancer is sufficient for obtaining the expected expression.

2 339 base pairs nucleic acid single linear DNA (genomic) not provided 1TGTTGGGGTT CAAATTTGAG CCCCAGCTGT TAGCCCTCTG CAAAGAAAAA AAAAAAAAAA 60AAAGAACAAA GGGCCTAGAT TTCCCTTCTG AGCCCCACCC TAAGATGAAG CCTCTTCTTT 120CAAGGGAGTG GGGTTGGGGT GGAGGCGGAT CCTGTCAGCT TTGCTCTCTC TGTGGCTGGC 180AGTTTCTCCA AAGGGTAACA GGTGTCAGCT GGCTGAGCCT AGGCTGAACC CTGAGACATG 240CTACCTCTGT CTTCTCATGG CTGGAGGCAG CCTTTGTAAG TCACAGAAAG TAGCTGAGGG 300GCTCTGGAAA AAAGACAGCC AGGGTGGAGG TAGATTGGT 339 1096 base pairs nucleicacid single linear DNA (genomic) not provided 2 CTGCAGCCTC AACTTCCTGGGCTCAAGCAA TCCTCCCACC TCGGCCTCCT AAAATACTGG 60 GATTATAGGC ATGAGCCACCACTCCCAGCA CCACTTTTTT CAGACTGGAA AAGAACACTC 120 ACATGTGCAT CTTTAAATGACACTTGGGCT GTGGTATGGA GAATGGCCAC CAGTGAGTAG 180 GCAGGAGCTG TTGTCCGAGCAAGGGCTGAT ATTGGCATCT TGGATTGGCA TGGTGGCAGT 240 AGTGGTAGTG CAGAGTGACTTGGGTAGATT TTGGAGCATT TAGAAGGTAC ATCCACAGGA 300 ACTGGTAAAT AAATACGTGGGAGAAGTTGG GTGAAGGGGG TGTCAAAGAT TACACCCAAT 360 TTATTTTGCT TGGGAAGTTGGTGGATGGTG AGCCCCTCAC TGAGTGAGAA GCCTGGAGAA 420 GCAGGTTTGG AGGGTGGTAGTATGCAGGTG GTATGCATAG TTGGGATGTG TGTTGAGTTT 480 GCTATGTCCG GTGAGCTTCCCAGTGGAGAT GTCCAATGGG CAGACGGATA CTCACATAGA 540 GAGTTCATGG TAGATTCGGGCTAGAGGAAA GCACCTGAGG CCTGGCCAGA GACGCCTAGA 600 GGAACAGAGC CTGGTTAACAGTCACTCCTG GTGTCTCAGA TATTCTCTGC TCAGCCCACG 660 CCCTCTCTTC CACACTGGGCCACCTATAAA GCCTCCACAG ATACCCCTGG GGCACCCACT 720 GGACACAATT GCCCTCAGGGCCCCAGAGCA AGGAGCTGTT TGTGGGCTTA CCACTGCTGT 780 TCCCATATGC CCCCAACTGCCTCCCACTTC TTTCCCCACA GCCTGGTCAG ACATGGCACT 840 ACCACTAATG GAATCTTTCTTGCCATCTTT TTCTTGCCGT TTAACAGTGG CAGTGACACT 900 TGACTCCTGA TTAAGCCTGATTCTGCTTAA CTTTTTCCCT TGACTTTGGC ATTTTCACTT 960 TGACATGTTC CCTGAGAGCCTGGGGGGTGG GGAACCAGCT CCAGCTGGTG ACGTTTGGGG 1020 CCGGCCCAGG CCTAGGGTGTGGAGGAGCCT TGCCATCGGG CTTCCTGTCT CTCTTCATTT 1080 AAGCACGACT CTGCAG 1096

What is claimed is:
 1. A method of causing selective expression of anucleic acid in mature T lymphocytes, comprising: (i) providing a vectorcomprising said nucleic acid operably linked to a CD4 enhancer sequenceand a CD4 promoter sequence, wherein the vector comprises, in the 5′→3′order: a sequence of a CD4 enhancer a sequence of a CD4 promoter thenucleic acid to be expressed, and a polyadenylation signal; and (ii)introducing said vector into hematopoietic cells in vitro or ex vivo,said introduction causing expression of said nucleic acid selectivelyinto T lymphcytes matured from said hematopoietic cells.
 2. The methodof claim 1, wherein the hematopoietic cells comprise hematopoietic stemcells.
 3. The method of claim 1, wherein the vector is a viral vector.4. The method of claim 1, wherein the nucleic acid encodes a toxicpolypeptide.
 5. The method of claim 1, wherein less than 1% ofnon-mature lymphocytes express the nucleic acid.
 6. The method of claim4, wherein the polypeptide is a thyrnidine kinase.
 7. The method ofclaim 1, wherein the vector comprises multiple copies of the CD4enhancer sequence.
 8. The method of claim 1, wherein the CD4 enhancercomprises at least one copy of the rnurine CD4 enhancer of SEQ ID No:1.9. The method of claim 1, wherein the CD4 promoter is selected from thegroup consisting of the sequence of SEQ ID No: 2, the sequence containedbetween nucleotides 579 and 1092 of SEQ ID No: 2 and the sequencecontained between nucleotides 912 and 1092 of SEQ ID No:2.
 10. Themethod of claim 3, wherein the viral vector is selected from the groupconsisting of a retroviral vector comprising two LTR sequences, anadenoviral vector and an adenoassociated virus vector.